Bactericide composition based on a mixture of bacteriophages for the control of black plague in plants or parts thereof, preferably the walnut, caused by xanthomonas arboricola pv. juglandis; preparation method and application

ABSTRACT

The invention relates to a bactericide composition based on bacteriophages for the control of black plague in plants or parts thereof, preferably walnuts, a preparation method and application. The invention provides methods for the isolation, propagation and application of bacteriophages against phytopathogens affecting trees/plants that are of commercial interest for their fruit, flowers etc., for the prevention, treatment or reduction of signs, in particular, for Xanthomonas a. pv juglandis in walnuts.

FIELD OF THE INVENTION

The present invention relates to a bactericidal composition based onbacteriophages for the control of the walnut blight in plants or partsthereof, preferably walnut; preparation method and application. Theinvention provides methods of isolation, propagation and application ofbacteriophages against phytopathogens affecting trees/plants ofcommercial interest for their fruits, flowers or others, forprophylaxis, treatment or reduction of signs, in particular, forXanthomonas a. pv juglandis in walnuts.

BACKGROUND

For producers of walnuts, and particularly for Chile—third LatinAmerican producer, the presence of Xanthomonas pv juglandis (Xaj), thecausative agent of the walnut blight of walnut (FIGS. 3A-3D) is a majorproblem. It is estimated that losses due to this disease, reach 50-80%in production (Chariot and Radix, 1993; Miller, 1934; Brandi et al,2014.).

The diagnosis of AKI is currently delayed when significant renal damagehas developed, making difficult to recover renal function, in part dueto the current lack of tools for early diagnosis.

Walnut blight is one of the most important diseases, and is widelydistributed in these crop areas (Loreti, 2001). Xanthomonas pv juglandiscauses severe damage to leaves, branches, buds, petioles, rachis,catkins, fruits among other tissues, and is considered one of the maincauses of performance decline in fruit and tree vigor (Belisario et al.,1999).

X. arboricola is a bacterial species associated with plants thatincludes strains responsible for the major diseases in stone fruit andnut strains. X. arboricola is divided into pathovars, some of which areclassified as quarantine organisms. The three most economicallyimportant species in pathovars are pruni pathovars, corylina andjuglandis responsible for bacterial spot of stone fruit trees, bacterialblight of hazelnut and walnut blight plague respectively. Recent studieshave shown that pathovars pruni, corylina and juglandis are closelyrelated phylogenetically (Fisecher et al., 2015).

Currently, the treatment available to control the walnut blight,consists mainly in agrochemicals based on copper. However, theseagrochemicals have noticeably lost its effectiveness, and even requiremore than 5 applications to control outbreaks of the disease(manufacturers recommended dose).

Compounds based on copper in its different formulations such asagrochemicals, are the main tool used to control plant diseases. Despiteits widespread use, its efficiency is increasingly challenged,especially due to the emergence of resistance in the phytopathogensmicroorganisms, and its important phytotoxicity.

This is how the effectiveness of treatment with copper-based productsfor control X. arboricola pv. juglandis has decreased over time, due tothe selection and increase of bacteria resistant to these agrochemicals,since bacteria have generated or acquired different resistancemechanisms.

This situation gets worse, by considering the evidence from differentscientific studies that suggest the transfer of copper resistance genesbetween bacteria. On the other hand, the excessive use of coppercompounds in the field has severe repercussions since this metalaccumulates on the floor in highly toxic waste for crops and theenvironment. In Chile, the control of the walnut blight, is based onapplications of copper products, which in a season can reach even up to10 or more applications, depending on location, weather conditions andinoculum pressure. This situation is explained by the number and poorapplication techniques, which have selected strains resistant to copper.

In fact, in 2007, the first local study was conducted (only Maule andMetropolitan regions), where the presence of X. arboricola pv juglandisresistant to Cu⁺² was determined (Esterio et al. 2007). Resistancelevels fluctuated between 8 and 64 μg/mL and a direct relation and closedependence between resistance level and copper selection pressure wereestablished.

Current results (in review), of the same inventors than the presentbactericide composition have established the presence of X. arboricolapv. juglandis in all sampled orchards of the four main nut producingregions in Chile (VI, VII, VIII and XIII). Furthermore, it has beenfound that more than 76% of all Xaj isolates are resistant toconcentrations higher than 16 μg/mL of Cu⁺² (FIG. 1).

This demonstrates that current management methods with compounds basedon copper, do not reach and effective control, causing the prevalence ofthis disease in most of the national walnut orchards. The situationdescribed above also applies to other plants such as stone fruit and notjust the walnut, where the combat, control or management of infectiondue to Xanthomonas arboricola pv juglandis presents the samedifficulties.

Arises, the need to develop more effective alternatives withoutphytotoxic risk and low environmental impact for the treatment of thewalnut blight. As a solution, the present invention suggests abactericidal composition based on a mixture of bacteriophages (phage),which is able to infect and kill specifically X. arboricola pv.juglandis, generating a control and effective protection to plants orparts thereof, including trees selected from the group consisting ofwalnuts and pits, and preferably, walnuts, against the walnut blight.

The major advantages of using a mixture of phages as a bactericide thatmay be mentioned are: its high specificity, its safety, its high rate ofreplication, the fast development of new formulations, and the fact ofbeing a natural product.

Specifically, in the agricultural field, additional advantages are: theunrestricted use, nor of entrance to orchard, neither of deficiencies,and it is feasible to use with cupric treatments. In addition, itsupports organic farming and integrated production.

Thus, the main objective of the present invention is a bactericidalcomposition based on bacteriophages to control the walnut blight orinfection Xanthomonas arboricola pv juglandis, in plants or partsthereof, including trees selected from the group consisting of walnutsand pits, and preferably walnuts.

It is yet another objective of the present invention, methods ofisolation, propagation and application of bacteriophage againstXanthomonas pv juglandis, for prevention, treatment or reduction ofsigns due to Xanthomonas pv juglandis on plants or parts thereof,including trees selected from the group consisting of walnuts and pits,and preferably walnuts. The composition is stabilized in a solution thatallows the viability and bactericidal activity of phages, and thusfavors the application thereof in the field.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Shows the percentage distribution Xanthomonas a. pv juglandisisolates resistant to different concentrations of copper sulfate.

FIGS. 2A and 2B: A: Shows lysis plates of bacteriophage ⊖4.1. B: Strainof 4.1 and 830 cultures of Xanthomonas a. pv juglandis in phagespresence Phages are capable of lysing completely bacterial cultures.

FIGS. 3A-3D: Displays fruits of walnut trees affected by walnut blight(Xanthomonas pv. juglandis).

FIG. 4: Shows a diagram of phages selection method of the presentcomposition.

FIG. 5: Digestion pattern of seven selected phage genomes. It wasperformed with the enzyme TaqI and 100 volts electrophoresed for onehour.

FIGS. 6A-6L: Electron microscopy of the seven bacteriophages from thepatent. Shows morphology of each of the bacteriophages.

FIG. 7: Scheme of bacteriophage isolation methodology used prior totheir characterization, with screening purposes for the mixture of thepresent composition

FIG. 8: Scheme of genomic characterization methodology of bacteriophagesused prior to the selection of the phages mixture of the presentcomposition (RFLP).

FIG. 9: Shows testing methodology of Xaj pathogenicity in tomato(Solanum lycopersicum).

FIG. 10: Shows test result of Xaj pathogenicity in snuff tree (Nicotianatabacum)

FIG. 11: Result of effectiveness of phages in controlling Xaj in unripewalnut fruits evaluation. It shows untreated group and treatment of aphage.

FIG. 12: Scheme for evaluating efficacy of phages in controlling Xaj, inunripe walnut fruits.

FIG. 13: Result effectiveness of phages in controlling Xaj in walnutleaves evaluation. Disease signs and re-isolation of the bacteria(laceration sheet method) are shown. A: Group Only with Xaj; B: Grouptreated with phage; C Medium control safety.

FIG. 14: Result of efficiency of the seven phages in controlling Xaj insnuff leaves (Nicotiana tabacum) evaluation. Disease signs andre-isolation of the bacteria (leaf infiltration method) are shown. A:Group Only with Xaj; B: Group treated with phage; C Medium controlsafety.

FIG. 15: Result of efficiency of the seven phages mixture in controllingXaj in walnut leaves evaluation. Disease signs and re-isolation of thebacteria (leaf infiltration method) are shown. A, B and C are differenttrees held in test.

FIG. 16: Shows the lytic cycle of a virulent bacteriophagecomprising: 1) fixation, 2) injection of the viral genome, 3) synthesis(transcription, translation, viral genome replication), 4) Assembly ofnew bacteriophages, and 5) release of new phage progeny (bacteriallysis).

FIG. 17: host range to the seven selected phages. Green boxes indicateno bacterial growth, little orange boxes indicate bacterial growth,yellow boxes indicate high bacterial growth, gray boxes indicate fullbacterial growth.

FIG. 18: Determination of temperature effect on the viability of thephage. The graph shows the inactivation degree inactivation of the sevenselected phages, for a temperature range between 4° C.-45° C.

FIG. 19: Determination of phages storage type effect. The graph showsthe degree of inactivation of the seven selected phages, when stored at−80° C. and 4° C.

FIG. 20: Determination of phages inactivation by exposure to copper ion.The graph shows the inactivation degree of the seven selected phage,when exposed to a solution of copper sulfate (64 mg/mL).

FIG. 21: Comparison of lysing capacity of the seven bacteriophages whenexposed to copper.

FIG. 22: Determination of phages inactivation by exposure to solarradiation. The graph depicts the degree of phages mixture inactivation,when exposed to direct sunlight.

DETAILED DESCRIPTION OF THE INVENTION

The present invention proposes a bactericidal composition based onbacteriophages, which is able to infect and kill specificallyXanthomonas arboricola pv juglandis (Xaj). The present invention alsoprovides methods of isolation, propagation and application ofbacteriophages against phytopathogens affecting trees/plants that are ofcommercial interest for their fruit, flowers etc., or others, forprevention, treatment or reduction of signs, in particular, forXanthomonas a. pv juglandis in walnuts.

Some advantages of the present composition are the following: its highspecificity, its safety, its high rate of replication, the fastdevelopment of new formulations, and the fact of being a naturalproduct. Additionally, and is compatible with the use of copper. Table 1briefly summarizes the advantages of the composition of the presentinvention based on the bactericidal copper compounds

TABLE 1 Main differences between use of bacteriophages and copper-basedagrochemicals such as treatment plants. Present composition Agrochemical(Cu) Specific Non specific Safe Toxic Autoreplicable Dilutable Naturalproduct Chemical product Without restrictions of use Restricted use(phytotoxic)

Specifically, in the agricultural field, additional advantages are: theunrestricted use, nor of entrance to orchard, neither of deficiencies,and it is feasible to use with cupric treatments. In addition, itsupports organic farming and integrated production.

Thus, the main objective of the present invention is a bactericidalcomposition based on bacteriophages to control the walnut blight inplants, especially walnuts.

It is yet another objective of the present invention the methods ofisolation, propagation and application of bacteriophage againstXanthomonas pv juglandis, for prevention, treatment or reduction ofsigns due to Xanthomonas pv juglandis on walnuts. The composition isstabilized in a solution that allows the viability and bactericidalactivity of phages, and thus favors the application thereof in thefield.

Bacteriophages are viruses that infect only bacteria, and thus areharmless to eukaryotic host cells (walnut, human, etc.). It is estimatedthat bacteriophages are the most abundant biological entity on theplanet with a ratio of 10 phage per bacterium. There are lyticbacteriophages that, after infecting and multiplying themselves in theirhost bacteria, destroy it (bacterial lysis), releasing a new progenybacteriophage, which is composed by hundreds of viral particles ready toinfect more bacteria (specific host) repeating the lytic cycle (FIG.16). Thus, the pathogenic bacteria is controlled, without affecting thebeneficial microbiota of trees nor leaving residues in the environment.

Results

The present composition comprises a mixture of phages is able to infectand kill, Xanthomonas a. pv juglandis, even resistant to currenttreatments (Cu⁺²) (FIGS. 2A and 2B) strains. Specifically, the presentbactericidal composition in one embodiment thereof, comprises sevenhighly lytic bacteriophages, characterized in atingentes theirproperties as bactericides. These bacteriophages are ⊖4.17 (depositnumber PCM F/00087), ⊖6 (deposit number PCM 00088), ⊖36 (deposit numberPCM 00089), ⊖M4-109a (deposit number PCM 00090), BM3 ⊖7-70 (depositnumber PCM 00092), 9M310-91a (deposit number PCM 00091), and ⊖M2-2(deposit number PCM 00093). The restriction pattern of the genomes ofthe phages is a quick and easy tool to differentiate and identify eachbacteriophage, this is shown for each of the phages in FIG. 5. Moreover,the seven bacteriophages were resistant to action of copper. The mainfeatures of these phages are shown in Table 2.

TABLE 2 Main characteristics and classification of selectedbacteriophages Genome Inacti- Diges- Size Mem- Phage vation RFLP Phagetion (pb) brane type copper Group Θ6 DNA 42288 Absence Podovirus No BΘ36 DNA 40834 Absence Podovirus No A Θ 4.1 DNA ND Absence Podovirus No AΘM4- DNA 39087 Absence Podovirus No A 109a ΘM2-2 DNA 38075 AbsenceSifovirus No C ΘM307- DNA 40302 Absence Podovirus No D 70a ΘM310- DNA NDAbsence Podovirus No B 91a

An important criterion for selecting bacteriophages agronomicapplication is having very low resistance in bacteria that infect. Thus,one can remove almost all of the pathogen, and the combination of themensures a very low probability that generate resistance to thecomposition. The percent resistance for each selected bacteriophage wasdetermined in their host strain and tabulated in Table 3.

TABLE 3 Determination of resistance percentage and information isolationBacteriophages % Xaj Type of sample Place of origin Phage resistanceHost of isolation (region) Q4.1 0.02 Q4.1 water Peñaflor (XIII) Q6 0.006 water Lonquén (XIII) Q36 12.00 36 water Lonquén (XIII) QM4-109a 0.09109a soil Amalia (VII) QM307-70a 0.00 70a water Porfirio (VIII) QM31-91a0.03 91a water La Perla (VIII) QM2-2 12.00 31 water Guitro (VI)

The lytic capacity was tested in vitro, on cultures of variousXanthomonas pv juglandis in solid and liquid media. Phages are able tocompletely lyse liquid cultures of the pathogenic bacteria, and generatesolid media plates completely transparent lysis (FIGS. 2A and 2B). Thisbactericidal composition in one of its embodiments comprises 7bacteriophages, which have a bactericidal action over 90% of the 87tested strains X. arboricola pv juglandis (Table 3), of which more than76% of the bacterial strains are resistant to different concentrationsof copper. These strains were isolated from different cultures fromwalnut nut main regions (regions VI, VII, VIII and XIII). This propertyof the present composition ensures a high probability of disease controlin different locations (Nocedales). Table 4 summarizes the host range ofbacteriophages 7 above, and the detail of each of them can be seen inFIG. 17.

TABLE 4 Summary of host range of 7 bacteriophages selected, tested withvarious strains of X. arboricola pv juglandis isolated from the mainproducing regions of nuts. The table shows the distribution of thedifferent isolates tested Xaj and percentage of coverage bactericidalcomposition shown phages. Group of Xaj Group of Xaj did not infected byphages infected by phages RM 17 4 5 1 VI 12 3 1 1 VII 18 4 1 1 VII 29 30 0 Culture 4 ND 0 0 Collection Total 80 14 7 3 Percentage ofbactericide coverage 91.95% Percentage without bactericide coverage 8.05%

Xaj strains that were virulent were chosen to determine this featuremodels and snuff tomatoes plants was used. In tomato surface it erodedleaves and branches, and bacteria (See FIG. 9) was added. To snuff theyleave with individual strains Xaj and mixtures thereof infiltrated (SeeFIG. 10). More virulent strains Xaj were chosen for evaluation testswith phages protection. To assess the ability to control Xaj withbacteriophages were performed using immature fruits of walnut (walnut),walnut leaves and leaves snuff. The results indicated that the groups ofuntreated fruits with bacteriophages, developed similar to the walnutblight, brown-black coloration signology in inoculation area andinternal necrotic (mesocarp) tissue, together with the recovery of thestrain Xaj inoculated. However, the groups treated with bacteriophagesome had lower degree of signology walnut blight, although generally thepresence of Xaj inoculated only in isolated cases and in lowconcentration was detected. Similar results were observed in trials withleaves, in the treated groups blackening was observed in puncture sitesand the presence of pathogenic bacteria in the area of infection, unlikethe groups treated with some of the bacteriophages of the presentinvention. All of the above according to detail provided in the Examplessection.

Therefore, the results showed that the groups not treated withbacteriophage generated small black spots in the puncture site, necroticendocarp tissue and the presence of bacteria in the area of infection,unlike the groups treated with the bacteriophages of the presentinvention. Thus, the present invention comprises native bacteriophages(bactericides) useful for conducting a treatment for Xanthomonas pvjuglandis control, the causative agent of the walnut blight. In thepresent composition it is stabilized said mixture in a solution thatallows the “viability” and bactericidal activity of phages, so, to favorits field application.

Finally, different media formulations who managed to stabilizebacteriophage in time and can be field applied safely and that these notFormulating the composition of antibacterial inactivate is aconcentrated form comprising in one of its embodiments, is evaluatedseven bacteriophages equivalent amounts of them (10⁹ PFU/mL).Antibacterial composition further comprises a buffer solution and/orstabilizing agents bacteriophages be so busy crops with safety tomaintain bactericidal activity. The buffer solution basically compriseone or more of the following components: NaCl, MGSC, Tris-HCl, gelatin,pH 7.5. As stabilizing agents may be used salts which maintains osmoticpressure and useful as cofactor phage absorption, among others.Furthermore, the antibacterial composition to has a high stability at atemperature range between 4° to 45° C.

EXAMPLES

The following examples are included and detailed to demonstrate thefeatures of the present invention.

Example 1

Means, isolation, and crops conditions for Xanthomonas arboricola pvjualandis strains

For proper isolation of bacteriophages, it is first necessary to isolatethe host bacteria. It is important to mention that “host bacteria or“host” refer to the bacteria that phages are able to infect, reproduceand lyse (kill).

To isolate Xaj, samples of leaves, fruits, and buds of walnuts withsymptoms of walnut blight were collected, coming from the main nutsproducer's regions of Chile (VI, VII, VIII and XIII) (FIG. 4).

The samples were processed using the method described by Moragrega etal. (2012). To obtain isolates of Xanthomonas arboricola pv juglandis, asegment of 1 cm² of infected tissue was macerated into 300 μL ofphysiological buffered water (buffer AFT) (0.14 M NaCl, 2.6 mMNaH₂P0₄.2H₂0 and 7.5 mM Na₂HP0₄.X 12H₂0). Some isolates were performedfrom the homogenized into solid medium B. King. (2% Protease Peptone,0.15% KaP0₄, 0.15% MgS0₂× TH₂0, 1% glycerol and 1.5% agar. PH 6.5-7.0).The plates were incubated at 28 C for a period of 72 to 120 hours.

Isolated colonies that showed the classic morphology Xaj (yellowcolonies, translucent, viscous and with smooth edges) were selected andidentified through amplification and subsequent sequencing of a 16Sribosomal gene segment. Basically, the 16S ribosomal gene of theisolates was amplified using universal primers 27F(5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′)(Weisburg et al, 1991). PCR conditions used were described by Trabal etal. (2012).

The amplificons of 16s ribosomal gene were digested by the enzyme Alu Iand run into electrophoresis in polyacrylamide gel at 7.5%. Thisgenerates a pattern of characteristic fragments for Xaj, which wassubsequently used for quick identification of these strains.

It was possible to obtain a strain collection of 87 Xanthomonasarboricola pv juglandis, characterized according to its level ofresistance to copper. The Xaj were preserved by cryopreservation inglycerol 50% v/v, frozen at −80 C, until its further use.

Getting Bacteriophages Example 2

Sample Processing, Isolation and Purification of Bacteriophages

Samples of soils under walnut trees and irrigation water from differentfarms were collected. To process the soil samples, a volume of SM buffer(NaCl 5.78 g; MgS0₄ 7H₂0 2 g; Tris 6.057 g gelatin 0.1 g for one literof water) was added to the sample, according to its weight, homogenizedand stirred for all night, then centrifuged at 6000 g for 15 minutes andfiltered through 0.2 micron filter pore. To process water sample, itcentrifuged at 6500 g for 15 minutes and filtered through 0.2 micronpore filter

10 mL of nutritious medium (beef extract 3 g, peptone 5 g for one literof distilled water) and 10 mL of samples were added to both filtrates(water and ground SM buffer). Each one was inoculated with 100 uL ofcultures from 5 different strains Xaj. It was incubated at 28° C. andstirred for 48 hours. Later, it was centrifuged at 6500 g for 15 minutesand the supernatant was filtered through 0.2 micron pore filter. Whenthe bacteria grow in the culture medium any phage present which caninfect her will proliferate altogether.

Phage detection was performed by lysis plaques (double agar method). Thephages in suspension are subsequently detected by their ability to lysebacteria and prevent their growth (FIG. 2A). Thus, when phages areplaced on a bacterial lawn of X. arboricola pv juglandis in a solidculture medium, clear circles are observed, called lysis plaques (PFU)or inhibition halo, generated by the multiplication of the phages from aviral particle, which reach more host bacteria preventing their growth.

For isolation and classification of phages of Xanthomonas arboricola pvjuglandis, a plate lysis was generated, well separated from others(single phage), assuming that is was initiated by the same viralparticle, so that all phages originated from this suspension come fromthe same viral particle and are practically identical (clone). To insureclonality the procedure was repeated three times.

In order to avoid the presence of non-virulent temperate phages,transparent lysis plates without bacterial growth inside will beselected, since it may be due to lysogenized bacteria Preparation ofphage stocks and conservation of phage collection. Phages werereproduced on plates with solid base nutritious (NB) medium, and thehost bacteria mixed with soft agar NB (0.7% agar) spread on the surface.After the soft agar turned into gel, 10 drops of 20 uL were added, andit was incubated at 28 C for 24-48 hours. Once bacteria lysis wasobserved due to infection of the phage, 20 ml_of SM buffer was added inthe plates and stirred for 6 hours. SM buffer containing phages wascollected, to be centrifuged and filtered (0.2 micron). These phagesuspensions were stored at 4° C. and at −70 C with 50% glycerol ordirectly. This method generates phage stocks with concentrations between10¹⁰-10¹¹ PFU/mL.

One phage collection of seven bacteriophages ⊖4.1 (deposit number PCMF/00087), ⊖6 (deposit number PCM F/00088), ⊖36 (deposit number PCMF/00089), ⊖M4-109a (deposit number PCM F/00090), ⊖M307-70a (depositnumber PCM F/00092), ⊖M310-91a (deposit number PCM F/00091), and ⊖M2-2(deposit number PCM F/00092) was obtained. The outline of this method isshown in FIG. 7.

Example 3

General Characterization of Bacteriophages

The general characterization of the selected phages was performed. Animportant component of the strategy of using phage as a bactericide, isthe use of phage mixtures that have different infection routes, toreduce the frequency of bacteria phage resistant strains. Phages withmany differences have usually different infection routes.

Determination of the size and nature of nucleic acids: nucleic acidsfrom bacteriophages was extracted and purified, for perform enzymatictests with deoxyribonuclease and ribonuclease, determining the genometype of bacteriophage. The size of the molecules will be estimated byagarose gel electrophoresis 0.4%, using high molecular weight standard.Seven bacteriophages were found to have DNA genomes and their sizes areshown in Table 2.

Presence of membrane. This was checked by sensitivity to organic solvent(chloroform 1% v/v). These agents affect the phages causing deficiencyin infection and therefore a decrease in their titles, A sensitive phagewill be considered, ie with membrane, when its title decreases at leastthree orders of magnitude. None of the bacteriophages presents membrane(Table 2).

Morphological structure. The size and shape of each bacteriophage wasdetermined through transmission electron microscopy. For this, thephages were purified and concentrated by CsCl concentration gradients,and then photographed (FIG. 6).

Example 4

Restriction of Fragment Length Profiles (RFLP)

For the differentiation of bacteriophages, a digestion of the sevenphage genomes, with the enzyme TaqI was performed, according to jmanufacturer's recommendations, for 3 hours. Followed by apolyacrylamide gel electrophoresis (7.5%) for 2 hours at 100 volts (FIG.8).

Bacteriophages were grouped into four restriction profiles, whichincluded: Group A: ⊖36, 9M4-109a and ⊖4.1 (deposit number PCM F/00087);group B: ⊖6 and ⊖M310-91a; group C: ⊖M2-2; and group D: ⊖M307-70a. Thisis useful in both measurements of its presence as well as in theprotection actions, since the genetic profile can be used as afingerprint (FIG. 5).

Example 5

Determination of Phage Host Range Against Xanthomonas arboricola pvjualandis

Infection specificity of the seven individually selected phage wasanalyzed through the double agar method. This test was performedincluding all the strains of the obtained strain collection (87isolates) in the step mentioned above. Three micro drops withconcentrated phage were added on a lawn of different X. arboricola pvjuglandis phage strains in order to test. The results obtained werepresented as: lysis plates with clear zone, plates with cloudy area, orwithout growth inhibition (FIG. 17). The information was tabulated inorder to identify the phages with lytic effect over the greatest numberof strains.

The percentage of bactericidal coverage (host range) among the 87strains Xaj was 91.95%.

Example 6

Determination of Resistance

the frequency of resistant bacteria was measured by counting the numberof colony forming units (CFU) persisting in the phage presence (afterinfection). Serial dilutions from a bacterial culture in exponentialgrowth were performed and were infected with a high multiplicity ofinfection (MOI) of phage. In parallel, uninfected controls wereperformed. The ratio between the number of CFU of the infected dilutionsversus controls uninfected was considered resistant frequency.

The seven phages had the following percentages of phage resistance: ⊖6and ⊖M307-70a an 0%; ⊖4.1 an 0.02%; ⊖M310-91a an 0.03%; ⊖M4-109a 0.09%;and phage ⊖36 and ⊖M2-2 12%. There was no resistance to the mixture ofthe seven bacteriophages.

Example 7

Evaluation of Phage Effectiveness on Xaj Infection Control.

a. Determination of Phage Protection Against Xaj in Fruits and Leaves(Individual Bacteriophages)

It consisted mainly in an artificial inoculation of the bacteria X. pvarboricola. juglandis on immature and asymptomatic walnut fruits andfree of walnut blight, which were used to evaluate the bactericidalactivity of bacteriophages. It was held under controlled environmentallaboratory conditions as described below (FIG. 12):

Immature fruits of nuts and leaves asymptomatic with walnut blight, werecollected to be used in in vivo inoculation trials of bacteriophagesagainst X. arboricola pv juglandis.

The fruit and leaves were disinfected by immersion in sodiumhypochlorite at 5% and subsequently washed with sterile distilled waterand dried at room temperature, before being infiltrated withbacteriophages (10⁹ PFU/mL) selected through injection, in theequatorial j zone of the fruits and on the leaves (in triplicate), andin turn inoculated with X. arboricola pv juglandis (10⁸ CFU/mL) in thedemarcated areas for protection. As positive infection and safetycontrols, fruits were inoculated with only pathogenic bacteria andbacteriophages. The fruits were incubated at 25° C. in sterile boxes, ata relative humidity of 90%, with photoperiods of 16 hours of light, for15 days. Incidence and severity (progression) of the infection ininoculated fruits was evaluated through a cross-section. The incidenceof infection was determined by the presence of necrosis signs around theinoculation site and the presence of X. arboricola pv juglandis in thefruit. The severity was determined by the percentage of necrosisgenerated in the fruit due to the infection. Results were comparedbetween groups of treated fruits with bacteriophages and untreated oneshe results indicated that the group of fruits infected with the strainof Xaj 27 and the use of only one phage (⊖M310-91a), presented a lightbrown color only in the area of infection, and no bacteria presence wasdetected in fruits. However, non-treated group with bacteriophage⊖M310-91a, presented blackening and necrotic tissue in over 75% of thefruit. In all cases, the pathogenic bacteria was recovered frominfection (FIG. 11). A similar result was obtained when the phage ⊖4.1(FIG. 12) was used.

For leaves the incidence and severity (progression) of infection wasevaluated by the presence of necrosis signs surrounding area ofinoculation and of X. arboricola pv juglandis presence. The severity wasdetermined by the advance distance generated from the injection point.Results were compared between groups of leaves treated withbacteriophage and untreated ones The results indicated that the tests oninfected leaves with strain Xaj 762 untreated with bacteriophagepresented black coloring in the puncture site and little progress of thedisease from the inoculation site (2-3 mm), bacteria was recovered fromall from inoculated leaves. When ⊖6 and ⊖4.1 bacteriophages (depositnumber PCM F/00087), are applied independently, a black-brown coloringis observed only in the inoculation site, also, no presence of the Xaj762 strain was detected. The brown coloring in the inoculated site wouldbe explained by the mechanical damage generated to get the bacteria topenetrate the tissue, because the controls presented the same signswithout presence of Xaj. A similar result was observed when only ⊖36phage was used. In the group inoculated only with the bacteria, littleprogression of the disease was observed, but a high Xaj load waspresent. (FIG. 13-A), not j when the ⊖36 phage and Xaj were used, thepresence of bacteria was only in some leaflets and with low load (FIG.13-B). There was no presence of the bacteria in controls (FIG. 13-C).

B. Determination of Phage Protection Against Xaj in Snuff Leaves(Mixture of Bacteriophages)

The snuff (Nicotiana tabacum) is a model plant that is used forvirulence studies of X. arboricola pv juglandis (Bandi, et al. 2014).The challenge of protecting against phage consisted in an artificialinoculation of the X. arboricola pv juglandis bacteria throughinfiltration by stomas in snuff leaves. The bactericidal activity ofbacteriophages on the pathogenic bacteria was evaluated this way. Thedesign of snuff plants test is described below:

Snuff plants (Nicotiana tabacum) germinated in laboratory conditionswere used, to make sure they are pathogens free.

Three groups were made: medium and phages safety controls; Xaj mixtureinfected group with; mixture of bacteria (strains Sag, Sag642 and Xaj10) and bacteriophage group ages. The groups were constituted by threesnuff plants, and three secondaries leaves of each one were used. Eachleaf was infiltrated in 4 sites. The mixture was of seven bacteriophageswas infiltrated and inoculated with a mixture of three virulent strainsof Xanthomonas arboricola pv juglandis infiltrated. Positive controls ofinfection and safety were inoculated only with pathogenic bacteria andbacteriophages respectively. The plants were left at room temperaturefor ten days.

Walnut blight signs development and presence of bacteria in theinfiltrated areas were analyzed. Results were compared between groups oftreated plants with bacteriophage and untreated ones. Re-isolation of X.pv arboricola juglandis was performed from infected leaves with diseasesigns, to determine the presence of the infection and if it was causedby the inoculated pathogenic bacteria.

The results indicated that the group that only had X. pv arboricolajuglandis strains presented disease signs in all plants (chlorotic,yellow and thinning areas in the infiltration site). Also, Xaj bacteriawas present on all sheets (FIG. 14-A). On the other hand, plants thatwere infiltrated with the mixture of bacteriophages and bacteria had nodisease signs. When the presence of Xaj was determined, it was onlyrecovered in some of infiltration sites and at a very low load, only thepresence of other bacteria not associated with the disease was observed(FIG. 14-B). Finally, no control presented disease signs nor presence ofpathogenic bacteria (FIG. 14-C).

C. Determination of Phage Protection Against Xaj in Walnut Leaves(Mixture of Bacteriophages)

It consisted mainly in an artificial inoculation of bacteria X. pvarboricola juglandis mixture through infiltration by stomas inasymptomatic and walnut blight free walnut leaves, which were used toevaluate the bactericidal activity of the bacteriophages. The test wascarried out in walnut trees as described below:

Free of Walnut Blight signs trees were choosen to be used in protectiontrials of bacteriophages against X. arboricola pv juglandis. Brancheswere selected in west direction in order to promote the bacteriainfection through management of environmental conditions. The thirdleaflet of walnut leaves was selected to be inoculated (infiltration).Four groups were formed: two medium and phages safety controls; infectedgroup only with a mixture of Sag, Sag642 and Xaj10 strains; and mixturesinfected group with the bacteria and seven bacteriophages. For eachgroup, two leaves by branch were infiltrated in triplicate, performingtwo infiltrations in the selected leaflets of each leaf. The mixture ofthe seven bacteriophages was infiltrated at a concentration of (10⁹PFU/mL total), and in turn inoculated with X. arboricola pv. juglandis(10⁸ CFU/mL). Positive controls of infection and safety were inoculatedonly with pathogenic bacteria and bacteriophages respectively. As safetycontrols bacteriophages mixture and medium were inoculated. The brancheswere wrapped with plastic bags covered with aluminum foil in order toachieve high humidity and heat, the test was performed for ten days.Walnut Blight development signs and the presence of bacteria in theinfiltrated areas were analyzed. Results were compared between groups oftreated and untreated fruits bacteriophages Re-isolation of X. pvarboricola juglandis was performed from infected leaves with diseasesigns to determine the presence of the infection and if it was caused bythe inoculated pathogenic bacteria.

The results indicated that in untreated group, infected with the mixtureof three Xaj strains (XajSag, Xaj10, Sag642), a damage between 75 to100% of necrosis in the infiltration site was observed; bacteria wererecovered from all the inoculated leaves (FIG. 15). When the mixture ofthe 7 bacteriophages (with treatment) was applied, a damage between 0 to25% was observed, and the bacteria was only present in some of theinfiltration sites in low concentration (FIG. 15). No control presenteddisease signs.

Example 8

Determination of Phage Inactivation by Exposure to DifferentTemperatures and Storage Types.

5 ml of each phage was incubated at temperatures of 4° C., 25° C., 30°C., 35° C., 40° C. and 45° C. for 15 min. Then, the title for each phagewas quantified at the mentioned the temperatures, the titles of sevenphages remained at the same log for the six tested temperatures (FIG.18).

The bacteriophage stocks were stored for four months at temperatures−80° C. and 4° C. After this time, the titles were compared with theoriginal concentrations of phages (FIG. 19). Both methods were optimalfor bacteriophages storage.

Example 9

Determination of Phage Inactivation by Exposure to Ionic, Compounds(Metallic and Nonmetallic) Used in Agrochemicals.

Compounds such as copper, iron, zinc, magnesium, manganese, boron,molybdenum, calcium, nitrogen, sulfur, phosphorus and potassium are usedas biocides for controlling diseases, as fertilizers or as stimulants inthe agricultural field.

Phages were exposed individually to an aqueous solution of coppersulfate (64 mg/mL). 100 uL of phage was mixed with 900 uL of the coppersolution and was incubated for 20 minutes. The, the phages were washedwith 1.5 mL of SM buffer through Amicon filtration (100K) and graduatesystem (FIG. 21). None of the phage was inactivated when exposed tocopper (FIG. 20).

Example 9

Determination of Phage Inactivation by Exposure to UV Photoprotection.

The mixture of bacteriophages (10¹⁰ PFU/mL of each one) was exposed todirect sunlight for 6 hours. Two preparations of phages mixtures wereadded separately to formulations F-30 and N-30, in order to evaluate thephotoprotective effect. The results are shown in FIG. 22.

1. Bactericidal composition for plants or parts thereof comprising at least one of the bacteriophages selected from the group consisting of bacteriophage deposit number PCM F/00087 (⊖4.1), bacteriophage deposit number PCM F/00088 (⊖6), bacteriophage deposit number PCM F/00089 (⊖36), bacteriophage deposit number PCM F/00090 (⊖M4-109a), bacteriophage deposit number PCM F/00092 (⊖M307-70a), bacteriophage as deposit number PCM F/00091 (⊖M310-91a) and bacteriophage deposit number PCM F/00092 (⊖M2-2) in solution.
 2. The bactericidal composition of claim 1 also comprising a buffer, a stabilizer more agents or a mixture thereof.
 3. The bactericidal composition of claim 1 wherein said buffer solution comprises one or more of the following components: NaCl, Mg8S₄, Tris-HCl, gelatin at pH 7.5.
 4. The bactericidal composition of claim 1 wherein said stabilizing agents are selected from salts that maintain the osmotic pressure and cofactors for phage absorption.
 5. The bactericidal composition of claim 1 having high stability at a temperature range between 4° C. to 45° C.
 6. The bactericidal composition of claim 1 comprising the bacteriophage deposit number PCM F/00087 (⊖4.1), bacteriophage deposit number PCM F/00088 (⊖6), bacteriophage deposit number PCM F/00089 (⊖36), bacteriophage deposit number PCM F/00090 (⊖M4-109a), bacteriophage deposit number PCM F 00092 (⊖M307-70a), bacteriophage deposit number PCM F/00091 (⊖M310-91a) and bacteriophage deposit number PCM F/00092 (⊖M2-2) in solution.
 7. The bactericidal formulation for plants or parts thereof comprising the composition of claim 1 in a concentrated form and stabilized in solution, wherein said bacteriophage deposit number PCM F/00087 (⊖4.1), deposit number PCM F/00088 (⊖6), deposit number PCM F/00089 (⊖36), deposit number PCM F/00090 (⊖M4-109a), deposit number PCM F/00092 (⊖M307-70a) deposit number PCM F/00091 (⊖M310-91a) or deposit number PCM F/00092 (⊖M2-2) is present in an amount equivalent to 10⁹ PFU/mL.
 8. Use of the bactericidal composition of claim 1 for the control and prevention of infection by Xanthomonas pv juglandis resistant to copper or copper resistant black plague, in plants or parts thereof.
 9. The use of claim 8 wherein said plant is a tree.
 10. The use of claim 8 wherein said tree is selected from a walnut or pit.
 11. The use of claim 8 wherein said plant is snuff.
 12. The method for treating and/or protecting plants from infection by Xanthomonas pv juglandis resistant to copper or copper resistant walnut blight, comprising the composition of claim 1 in the leaves, fruits and buds of the plants.
 13. The method of claim 12 wherein said plant is a walnut.
 14. The method of claim 12 wherein said plant is a tree.
 15. The method of claim 14 wherein said tree is a walnut or pit.
 16. The method of claim 12 wherein said plant is snuff.
 17. Method for preparing the composition of claim 1 comprising: a) to isolate and to identify bacteria Xanthomonas arboricola pv juglandis when collecting samples of leaves, fruits and nut buds infected with said bacteria; to isolate this one from an infected tissue segment and to incubate plates at room temperature; to identify by amplification techniques and standards PCR sequencing, a 16S ribosomal gene segment amplifying said isolated with universal primers SEQ ID NOs. 1 and 2 to then digest amplificons of 16S ribosomal gene with the enzyme Alu I and through electrophoresis in polyacrylamide gel, to generate a characteristic fragment pattern of copper resistance for Xanthomonas arboricola pv juglandis, and to prepare a culture with said bacteria, b) to isolate and to purify bacteriophages against Xanthomonas arboricola pv juglandis while collecting samples of soils under walnut trees, including soil samples and also irrigation wafer samples from the vicinity of walnuts frees where soil samples were collected; to process the soil and water samples by centrifuging and filtering, for then adding to said filtered, a nutritious medium comprising beef extract and peptone in distilled water; c) to inoculate at room temperature while stirring, the said filtered with Xanthomonas arboricola pv juglandis cultures previously prepared in step a), for then centrifuging and filtering the supernatant; to detect phages that infect Xanthomonas arboricola pv juglandis in a solid culture medium, by observing light circles or lysis plaques or inhibition halos; d) to isolate and to classify Xanthomonas arboricola pv juglandis phages, and to reproduce them on plates with a nutritious solid medium from which the phages are then recovered in a suspension, once confirmed that they cause lysis of the said bacteria, and the suspension is stored at a temperature between 4° C. and −70° C. into glycerol, generating a composition comprising at least one of the phages deposit number PCM F/00087 (⊖4.1), deposit number PCM F/00088 (⊖6), deposit number PCM F/00089 (⊖36), deposit number PCM F/00090 (⊖M4-109a), deposit number PCM F/00092 (⊖M307-70a) deposit number PCM F/00091 (⊖M310-91a) or deposit number PCM F/00092 (⊖M2-2) with concentrations between 10¹⁰-10¹¹ PFU/mL.
 18. (canceled)
 19. The method of claim 17 comprising classifying phage according to its size which is determined by high molecular weight agarose gel electrophoresis; determining the type of genome to extract and purify nucleic acids thereof, and then perform enzymatic assays with deoxyribonuclease and ribonuclease to differentiate them; determining the presence in these membrane presence after exposure to an organic solvent including chloroform; determining its size and shape by transmission electron microscopy after purified and concentrated prior to microscopy.
 20. The method of claim 19 wherein said phages differentiation is performed by genomes digestion with enzyme TaqI followed by polyaerylamide gel electrophoresis. 